Notebook:Federico Castro M/2008/01/20

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Proposed Procedure

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• Status: Pending for approval
• Issues: None

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I want to recover 2 biobricks and later assemble them in order to create intermediate part for the construction for the diffusible signal oscillator (model 3). This part is by itself an inverter, it is not available at the registry. This could be the first part actually assembled by Mexico and the first part to reach the registry, if we hurry up this part may even be available at the iGEM kit plates 2008. The biobricks are oscillators dependent on quorum sensing signals such as the one I want to assemble. I await for approval for this procedure.

Biobricks to be extracted

• <bbpart>BBa_I0462</bbpart> available at well 11O at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3
• <bbpart>BBa_R0063</bbpart> available at well 13K at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3

The following protocol is proposed for biobrick recovery from the iGEM page [1]

1. Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick™-standard part that you want
2. Add 15 uL of diH2O (deionized water)
3. Take 1uL DNA and transform into your desired competent cells, plate out on a plate with the correct antibiotic* and grow overnight. Your goal here is to obtain single colonies.

I intend to use the following protocol for transforming the biobricks into competent cells.

1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
3. Incubate on ice for 30 minutes
   • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
4. Incubate cells for 30 seconds at 42°C.
5. Incubate cells on ice for 2 min.
6. Add 4 volumes of room temperature SOC (not critical)
7. Incubate for 1 hour at 37°C on shaker.
   • Note: Can also save some time here by reducing incubation to ~45 min.
   • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37°C.

Before the procedure I would need to prepare solid plates with Kanamicyn (all of them have resistance to Kanamycin). It seems that the proper concentration of Kanamicyn is 50 μg/mL.[2]

Latter we should extract the plasmids and do the apropiate cuts with endonucleases and the ligations. Since R0063 is very small we should not separate it from the plasmid, therefore we should use EcoRI instead of PstI.